The recent identification of arachidonlyethanolamide (AEA, also called anandamide) as an endogenous substance from brain that has activity as a ligand of the cannabinoid receptor is very exciting, however, many basic questions concerning the role of AEA as a signalling molecule in the brain must be answered. The overall hypothesis to be tested by this proposal is that AEA is produced and metabolized by the brain and has the characteristics of an endogenous ligand of the cannabinoid receptor. There are three specific aims of the proposal: (1) To test the hypothesis that AEA is inactivated in the brain by a combination of catabolism and uptake into cells. In specific, we will characterize the uptake of AEA by neuronal and glial cells and determine whether it is sodium and energy dependent and whether analogs of AEA compete for the process; second, we will determine the cellular distribution of AEA hydrolysis and tits structural requirements; (2) To test the hypothesis that AEA is synthesized by cells of the brain, we will determine whether AEA concentrations are increased by agents that increase the release of free arachidonic acid in brain tissue or if AEA is released from phospholipids as a result of phospholipase D activation; we will determine whether AEA is released by conditions that release other neurotransmitters; (3) To test the hypothesis that varying the structure of the ethanolamine portion of AEA is well result in analogs that have different binding characteristics for the cannabiniod receptor as well as altered susceptability to hydrolysis and uptake. The scientific personnel involved in the studies outlined in this proposal have considerable experience and expertise in two pertinent areas: the neurochemistry of the cannabinoids and the chemistry and biochemistry of arachidonic acid metabolites (eicosanoids). The methods that will be used to carry out the studies in this proposal include procedures for the separation and quantification of labeled and endogenous AEA, radioligand binding studies and studies of the efficacy of cannabinoid-like compounds and superfusion studies for the determination of AEA release. We will use several in vitro preparations in these studies, including brain slices and neuronal and glial cells in primary cultaure for catabolism, synthesis and release studies and membrane preparations for determination of binding to the cannabinoid receptor and cells stably transfected with the cannabinoid receptor to study inhibition of adenylyl cyclase activity. We will also assay cannabinoid activity using the mouse vas deferens contraction assay and measurement of effects on rectal temperature. We anticipate that the successful completion of the studies outlined in this proposal will provide valuable insight into the possible role of AEA as a signalling molecule in the brain. In addition, we will develop several tools that will be valuable additions to the cannabinoid field in general, including a sensitive assay for AEA and a series of active and inactive analogs.